1. Volume 14, Issue 2, Pages 347-354 (January 2016)
Differential Thiol-Based Switches Jump-Start Vibrio cholerae Pathogenesis 
Zhi Liu, Hui Wang, Zhigang Zhou, Nawar Naseer, Fu Xiang, Biao Kan, Mark Goulian, Jun Zhu 
Cell Reports 
Volume 14, Issue 2, Pages (January 2016)
DOI: /j.celrep
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2. Cell Reports 2016 14, 347-354DOI: (10.1016/j.celrep.2015.12.038)
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3. Figure 1
Tn-Seq Identification of OhrR as a Redox-Dependent Virulence Activator
(A) Schematic depiction of the Tn-seq screen.
(B) Volcano plot of Tn-seq results. Total output and input mapped read counts of 296 defined Tn mutants from infant mice infected with aerobic or microaerobic cultures of V. cholerae were normalized against p value. Each spot represents an identified TnFGL3 mutant. x axis is the average ratio of output reads/input reads (inocula from O2−-grown cultures) divided by the average ratio of output reads/input reads (from O2+-grown cultures) of each mutant.
See Figures S1–S3 for effect of AphB on mouse colonization and ΔohrR growth rate and Table S1 for primers used for Tn-seq.
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4. Figure 2 OhrR Activation of Virulence during Oxic-to-Anoxic Transition
(A) Effects of OhrR on virulence gene transcription. Wild-type and ΔohrR containing promoter-luxCDABE transcriptional fusion reporter plasmids of virulence gene tcpA and other major virulence regulator genes were grown aerobically and then incubated in AKI at 37°C for 1 hr without shaking. Luminescence was then measured and normalized against OD600. Expression in ΔohrR was compared with that in wild-type. Results are the means and SD of three independent experiments.
(B) OhrR effect on tcpP expression during O2+→O2− transition. ΔohrR, wild-type, and Ptac-ohrR (ohrRC) strains containing a PtcpP-luxCDABE were grown as above. tcpP expression was calculated by normalizing luminescence against OD600. Results are the means and SD of three independent experiments.
(C) Sequence alignment of the OhrR binding sites at the tcpP and ohrA promoters in V. cholerae (Vc) and the ohrA promoter in Xanthomonas campestris (Xc), Bacillus subtilis (Bs), and Agrobacterium tumefaciens (At). Conserved sequences of OhrR binding site are highlighted.
(D) Gel-shift assays using purified OhrR-His6 and tcpP promoter. 0.1 ng tcpP promoter DNA in a buffer with or without 1 mM DTT were incubated with different concentrations of OhrR as indicated and the samples were size-fractionated using polyacrylamide gels. See Figure S4 for AphB gel shift.
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5. Figure 3
Effects of Cysteine Residues of OhrR and AphB on tcpP Expression
ΔohrR mutants containing Ptac-ohrR (wild-type and cysteine mutant derivatives) plasmids (A) and ΔaphB mutants containing PBAD-aphB (wild-type and cysteine mutant derivatives) plasmids (B) were grown aerobically in LB containing 0.1 mM IPTG or 0.1% arabinose at 37°C until early-log phase. RNA was extracted and qRT-PCR was performed to determine tcpP transcription levels. recA was used as an internal control for each sample. Fold changes of tcpP expression in different strains were shown as means and SD of three independent experiments. ∗p < 0.01, Student’s t test. See Figures S5A and S5B for ohrRC128S colonization data.
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6. Figure 4
Dynamics of OhrR and AphB Reduction during Oxic-to-Anoxic Transition
(A) AphB and OhrR conformational changes in response to DTT by using ANS binding assays (Matulis et al., 1999). The function of DTT concentration on AphB or OhrR structural changes was calculated using the following formula: Δ482 nm (FR) = (Rcertain λ482nm − control λ482nm)/(Rmax λ482nm − control λ482nm) × 100, where FR indicates the function of reagent (DTT), Rcertain indicates the certain concentration of DTT used in experiment, control indicates without DTT, and Rmax indicates 1,000 μM DTT. Results are means and SD of three independent experiments.
(B and C) Thiol-trapping experiments. Early-log aerobically grown cultures of ΔaphBΔohrR mutants carrying Ptac-ohrR-FLAG and PBAD-aphB plasmids were shifted to the anaerobic chamber. At the time points indicated, trichloroacetic acid was added. The precipitated proteins were reacted with mPEG-maleimide and detected by SDS-PAGE and western blots. Representative images from three independent experiments are shown.
(D) The percentage of reduced proteins at different time points was quantified using ImageQuant software (Life Sciences).
∗p < See Figure S6 for AphB reduction rate in ΔohrR mutants.
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7. Figure 5 Effect of Sensitive Reduction of OhrR on Virulence In Vitro
(A) OhrR effects on tcpP during the transition from oxic to anoxic environments. Early-log aerobically grown cultures of wild-type, ΔohrR, aphBC235S, ohrRC128S, ΔaphB, and ΔaphBΔohrR mutants were reinoculated into AKI medium and incubated at 37°C in an anaerobic chamber. Samples were collected at the time points indicated for RNA purification and real-time qPCR examination of tcpP. The induction of tcpP was normalized to wild-type at 0 min. Results are the means and SD of three independent experiments. ∗p < 0.01 (compared to wild-type at the same time point).
(B) OhrR effects on downstream virulence gene tcpA. Early-log aerobically grown cultures of wild-type and ΔohrR containing PtcpA-luxCDABE plasmids were reinoculated into AKI medium and incubated at 37°C in an anaerobic chamber. Samples were taken out of the anaerobic chamber at different time points and luminescence was measured.
(C) Anaerobically grown ΔohrR mutants do not affect tcpA expression. Overnight cultures of wild-type and ΔohrR mutants containing a PtcpA-luxCDABE plasmid were refreshed in LB medium and were incubated at 37°C anaerobically to optical density 600 (OD600) ∼0.5. The cultures were spun down and resuspended with fresh AKI media and incubated at 37°C anaerobically. Luminescence and OD600 were then measured. The tcpA expression was calculated by normalizing luminescence against OD600. Results are the means and SD of three independent experiments.
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8. Figure 6 Effect of Sensitive Reduction of OhrR on Virulence In Vivo
(A and B) tcpP (A) and downstream tcpA (B) expression kinetics of wild-type and ΔohrR in vivo. Wild-type and ΔohrR harboring lacZ::res1-tet-res1 and tcpP::tnpR or tcpA::tnpR (Lee et al., 1999) were grown aerobically at 37°C in LB before inoculation into infant mice. At the time points indicated, bacteria were recovered from mice and examined for resolving of tetracycline-resistance cassettes. For each data point, over 300 colonies were examined. The percentage of Tet-sensitive (TetS) CFU was then calculated. ∗p < 0.01.
(C) ΔohrR and ΔaphBΔohrR colonization. Wild-type (WT) and mutants were grown microaerobically (O2−) or aerobically (O2+) to mid-log. Colonized bacteria were enumerated and competitive index (CI) was calculated by normalizing the output ratio of mutant/WT with the input ratio of mutant/WT. Horizontal lines represent average CI. ∗p < 0.01.
(D) Working model. AphB and OhrR respond to redox-potential changes differently. This results in differential regulation of tcpP so that promptly reduced OhrR jump-starts virulence during the transition from aquatic environments into the host. See Figure S5C for additional data on effect of OhrR on colonization.
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